The objectives of the proposed research are the following: (1) To characterize in detail the different levels of the subunit structure of Lumbricus hemoglobin. This will include determination of the molecular weights and number of copies of the constituent polypeptide chains and of their arrangement in the subunits making up the native molecule. (2) To compare the subunit structure of Lumbricus hemoglobin with the subunit structure of other annelid hemoglobins, namely those of Arenicola, Tubifex and Limnodrilus. (3) To determine the distribution of heme among the subunits of the native Lumbricus and Arenicola hemoglobins and the role of the heme-carrying subunits in the function of the native hemoglobin molecules. The subunit structure of hemoglobins will be determined using gel filtration and polyacrylamide gel electrophoresis in the presence of detergents and at alkaline pH. The number of copies of the isolated subunits will be determined by quantitative polyacrylamide gel electrophoresis. Identification of the constituent polypeptide chains will be completed using end group analysis together with stepwise Edman degradation whenever necessary. Crosslinking of the subunits in the intact molecule will be carried out using bifunctional reagents followed by identification of the crosslinked subunits using previously developed methods of dissociation. The resulting topology of the subunits will be compared with the proposed model of the quaternary structure of Lumbricus hemoglobin. The function of the heme-containing subunits will be studied by measurement of oxygenation of the individual subunits and reconstituted aggregates thereof in comparison with that of the native molecule.